PROJECTSUMMARY Epigenetic regulators represent a new frontier in drug discovery and are rapidly attracting pharmaceutical interests due to their remarkable ?druggability? and strong connection with human diseases (e.g., metabolic syndromes, neurodegenerative disorders, and cancers). To date, the epigenome consists of over 300 known proteins. However, very few epigenetic modifiers are being pursued in drug development programsduetoourlimitedunderstandingofwhatproteinstheseenzymesregulate.Thisnarrowsaturationof the epigenetic ?drug arena? is fueled by a dearth of tools to systematically identify and characterize potential targets.Toaidinthediscoveryofnovelrelationshipsbetweenenzymesandtheproteinsthattheytarget,wewill commercialize high complexity oriented peptide array libraries, (OPALs). In Phase I studies, we synthesized OPAL libraries, comprising a core arginine (R-OPAL) or lysine (K-OPAL) residue with flanking degenerate sequences (+/- 3 or 4 amino acids in both N- and C-terminal directions; Figure 1) and demonstrated that OPALs are powerful tools to systematically characterize target motifs of writer enzymes. Our focus here is to developOPALstoexamine?writer?enzymesthatmodifylysineorarginineresidues.InPhaseII,EpiCypherwill scaleupsynthesisoftheunmodifiedK-OPALandR-OPALpeptidelibrariesandoptimizeahigh-throughput96- wellassayformatforvalidationusingmethyltransferaseassaysdevelopedinthePhaseIstudies(Aim1).We will also vastly expand the commercial utility of our discovery platform on two fronts: 1) developing a non- radioactivedetectionsystemandauser-friendlydataanalysispipelineestablishing(Aim2)?and2)expanding ourK-OPALplatformtoanalyzelysine-acylationwriterenzymes(Aim3).Sevenadditionallysine-modifiedacyl group post-translational modifications (of which acetylation is a founding member) have been recently identified(e.g.,crotonylation,butyrylation,orpropionylation),whichfunctiontogetherwithlysineacetylationto regulate gene activation. Given the well-described role of histone acetylation in disease, these new acylation PTMs represent an exciting new field of epigenetic study that has great drug development potential. The innovative discovery platform described herein provides the first comprehensive screening tool to study epigenetic ?writer? enzymes. EpiCypher has assembled a strong scientific team consisting of pioneers in the field of epigenetic regulation (Drs. Mark Bedford and Scott Rothbart) and detection chemistry (Dr. Marcey Waters) to develop a robust, versatile, and user-friendly discovery platform that will have a powerful translationalimpactonepigeneticresearchandtherapeuticdevelopment.